The present invention relates generally to the development and use of a gene encoding a recombinant protein useful as a multivalent and multistage malaria vaccine and more specifically relates to a recombinant antigenic protein useful for preventing or treating P. falciparum malarial infections.
Malaria is a parasitic infection known to be produced by the Plasmodium species P. falciparum, P. vivax, P. ovale, and P. malariae. Humans become infected following the bite of an infected anopheline mosquito, the host of the malarial parasite. Malaria occasionally occurs in humans following a blood transfusion or subsequent to needle-sharing practices as used by drug addicts.
When an infected anopheline mosquito bites an individual, sporozoites present in the mosquito""s saliva are injected into the blood. The initial development of parasites occurs in the liver and is referred to as the liver stage, or the hepatic or exoerythrocytic phase. In this phase, the sporozoite grows and divides, producing numerous tissue merozoites. These merozoites rupture the hepatocyte and enter the circulation. Some merozoites attach to receptor sites on red blood cells, penetrate the plasmalemma and begin a development phase known as the asexual, erythrocytic cycle. Within the erythrocyte, the parasite is recognizable as a ring-stage trophozoite. These trophozoites enlarge, divide and attain the schizont stage. After successive nuclear divisions, the erythrocyte ruptures, releasing merozoites which attach to receptors on erythrocytes and thus begin another erythrocytic cycle. In P. vivax and P. ovale, hepatic parasites persist and may lead to a relapse of the disease months or years after the initial infection.
Some merozoites that enter red blood cells develop into male and female gametocytes. When a mosquito bites an individual possessing erythrocytic gametocytes and ingests them, the gametocytes are fertilized in the stomach of the mosquito and mature into sporozoites that migrate to the salivary glands. In this manner, the mosquito is capable of biting and infecting another individual.
Malaria is one of the most common infections of humans. It is estimated that malaria parasites cause about 300-500 million illnesses and 3 million deaths each year. Most of the severe morbidity and mortality occurs in children and pregnant women, and is caused by P. falciparum (World Health Organization (1989) Weekly Epidemiol. Res. 32, 241-247). While sub-Saharan Africa accounts for more that 90% of these cases, malaria is a serious public health problem for nonimmune individuals and servicemen and servicewomen traveling through and/or stationed in malarious regions of the world. Clinical manifestations of malarial infection which may occur include blackwater fever, cerebral malaria, respiratory failure, hepatic necrosis, and occlusion of myocardial capillaries. An effective vaccine that prevents or reduces infection and minimizes morbidity and mortality will be a very useful tool for the control and prevention of this disease.
The development of an effective malaria vaccine represents one of the most promising approaches for providing cost-effective intervention along with other control measures currently available. Over the last decade there has been considerable progress in the understanding of immune mechanisms involved in protection against parasites and clinical illness. Several malarial antigens have been identified for their ability to confer protection against malaria.
Three main types of malarial vaccines are currently under research and development, based on stages of the parasite""s life cycle. The three vaccines are generally directed to the following stages in the life cycle: 1) blood stage, including the asexual blood stage; 2) the sexual stages; and 3) preerythrocytic stages, including the liver stage. Antigens from each of these stages have been identified, the most promising being antigens from the following proteins: circumsporozoite protein (CSP) and SSP-2 protein of the sporozoite stage; the antigen (LSA-1) of the liver stage; the merozoite surface protein-1 (MSP-1), merozoite surface protein-2 (MSP-2), the rhoptry associated protein-1 and -2 (RAP-1 and RAP-2), the erythrocyte binding antigen-175 (EBA-175) and apical membrane antigen-1 (AMA-1) of the asexual blood stage; and the ookinete antigen Pfs 25 and the gamete specific antigen Pfg27 of the sexual stage.
Therefore, what is needed is a single vaccine that provides immunogenicity or confers immunity against various stages in the life cycle of the malarial parasite, particularly P. falciparum, to treat, minimize or prevent infection and reduce associated morbidity and mortality.
An antigenic recombinant protein, method of making the protein, genetic construct encoding the protein, antibodies to the protein, pharmaceutical composition containing the protein, and a method for the treatment, prevention or reduction of malarial infection by administering the protein to a human or animal are provided. The protein and anti-protein antibodies are useful as research or diagnostic reagents for the detection of the Plasmodium species P. falciparum in a biological sample. When administered to human or nonhuman animals, the protein is effective against malaria by conferring immunogenicity or immunity against various stages in the life cycle of the malarial parasite P. falciparum. 
The antigenic recombinant protein is prepared by constructing a gene that encodes stage-specific antigenic determinants. The gene is added to a vector and is then expressed in a suitable expression system, such as a baculovirus system, to produce a single protein that confers immunity against different stages in the malarial life cycle of P. falciparum, or provides immunogenicity against epitopes from different stages in the life cycle of the parasite. In the present invention, these stages are the sporozoite stage, the liver stage, the blood stage and the sexual stage (also known as the gametocyte stage). By using a combination of antigens or epitopes derived from different stages in the life cycle of a malarial parasite, the protein constitutes an efficacious, cost-effective, and sustainable multicomponent vaccine for use in malaria control programs. The protein, in a pharmaceutically acceptable carrier, specifically provides a multivalent and multistage vaccine for malaria caused by the parasite P. falciparum. 
The immunogenic regions of the various stage-specific antigens of P. falciparum used to construct the gene encoding the antigenic recombinant protein are selected based on immune response studies in clinically immune adults and in vitro immune response studies using peptides and/or antibody reagents. The resulting synthetic gene is sequence-confirmed and expressed in a baculovirus expression system. The preferred antigenic fragments used to make the coding sequences used in construction of the gene are shown in Table 1. The nucleotide sequence of the preferred gene is shown in SEQ ID NO:1. The amino acid sequence of the preferred recombinant protein encoded by the gene, referred to herein as CDC/NIIMALVAC-1, is shown in SEQ ID NO. 2. The recombinant protein in a pharmaceutically acceptable carrier is useful as a multivalent, multistage vaccine for P. falciparum malaria.
The vaccine described herein is a cost-effective, health-promoting intervention for controlling, preventing or treating the incidence of malaria. The vaccine is useful for reducing sickness, morbidity, mortality and the cost of medical care throughout the world. Similarly, the vaccine is useful for preventing or reducing malarial infection in U.S. citizens and military personnel traveling or living in regions of the world where malaria is present. The vaccine is also useful for decreasing the severity of the malarial disease process when administered after initial infection with P. falciparum. 
The vaccine is immunogenic as confirmed by its ability to elicit immune responses against both the vaccine protein and the P. falciparum parasite. In vitro tests of protection conferred by the vaccine against blood stage malarial parasites reveal that antibodies against this vaccine inhibit reproductive growth of P. falciparum. The vaccine also induces multiple layers of immunity to different stages in the parasitic life cycle of P. falciparum. 
It is therefore an object of the present invention to provide a multivalent, multistage vaccine against malaria.
Another object of the present invention is to provide a multivalent, multistage vaccine against malaria caused by P. falciparum. 
Yet another object of the present invention is to provide a vaccine against malaria that is effective in inhibiting reproductive growth of the parasite within a human or animal after initial infection.
Still another object of the present invention is to provide a gene useful as a DNA vaccine, or for production of a recombinant protein in various expression systems, the recombinant protein containing antigenic epitopes to various stages of a malarial Plasmodium species, particularly P. falciparum. 
Another object of the present invention is to provide a vector comprising a gene useful for production of a recombinant protein in various expression systems, the protein containing antigenic epitopes to various stages of a malarial Plasmodium species, particularly P. falciparum. This vector may be used for a variety of purposes including but not limited to administration to animals and humans, and for transfection of cells.
Yet another object of the present invention is to provide a recombinant protein containing antigenic epitopes to various stages of P. falciparum that may be used as a reagent or a multivalent, multistage antimalarial vaccine.
It is another object of the present invention to provide a method for conferring immunity against different stages in the life cycle of the malarial parasite, P. falciparum. 
Another object of the present invention is to provide a method of vaccination against malaria caused by infection with P. falciparum. 
It is another object of the present invention to provide a method to reduce morbidity and mortality associated with malarial infection by preventing malarial infection and also ameliorating the morbidity and mortality associated with malaria after initial infection with the parasite, P. falciparum. 
Another object of the present invention is to provide antibodies against a recombinant protein containing antigenic epitopes to various stages of P. falciparum, that are useful as research or diagnostic reagents for the detection and measurement of P. falciparum in a biological sample.
Yet another object of the present invention is to provide a more effective, simpler and economical vaccine for conferring immunogenicity to different stages in the life cycle of P. falciparum than prior art vaccines.
An advantage of the anti-malaria vaccine of the present invention is that it confers immunogenicity against several stages or all stages in the life cycle of P. falciparum with administration of a single vaccine, as opposed to multiple injections for each stage of the life cycle of the parasite.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments.